Extraction of heterologous insoluble proteins from bacteria

Abstract

An insoluble mammalian protein is extracted from transformed bacteria expressing the mammalian protein while avoiding irreversible insolubilization of bacterial host proteins by homogenizing the fermentation broth, centrifuging the homogenized broth and removing the supernatant liquid for the inclusion body containing pellet. In another embodiment, the pH of the homogenized broth is adjusted to 2.0 prior to centrifugation. The acidified broth is then centrifuged, and the pellet is resuspended in buffer, homogenized again, and the inclusion body is isolated by centrifugation.

What is claimed is: 
     
       1. A method of extracting a heterologous protein from bacteria expressing said heterologous protein comprising the following steps: (a) fermenting the bacteria in a fermentation broth;   (b) disrupting the bacteria contained in the fermentation broth;   (c) cooling or maintaining the fermentation broth at a temperature of between about 0°-15° C.;   (d) adding an acid to the fermentation broth such that the fermentation broth obtains a pH of about 2.0;   (e) incubating the acidified fermentation broth at a temperature of between 0°-15° C. so as to kill any remaining undisrupted bacteria;   (f) centrifuging the fermentation broth so as to obtain an inclusion body pellet and a supernatant liquid;   (g) removing the supernatant liquid from the inclusion body pellet;   (h) suspending the inclusion body pellet in a buffer so as to obtain a suspension;   (i) adding a pH adjusting solution to the suspension so that the supspension obtains a pH of between 6-9;   (j) disrupting suspended solids contained within the suspension of step (i);   (k) centrifuging the suspension so as to produce an inclusion body pellet containing the heterologous protein and a supernatant liquid; and   (l) removing the supernatant liquid so as to obtain isolated inclusion bodies containing heterologous protein.   
     
     
       2. The method of claim 1 wherein the acidified fermentation broth is maintained at a temperature of 0°-25° C. 
     
     
       3. The method of claim 1 wherein the acidified fermentation broth of step (d) is incubated for about an hour prior to centrifugation at a temperature between 0°-15° C. 
     
     
       4. The method of claim 1 wherein the acid is selected from the group consisting of phosphoric acid, nitric acid, hydrochloric acid and sulfuric acid. 
     
     
       5. A method of extracting a heterologous protein from bacteria expressing said heterologous protein consisting essentially of the following steps: (a) fermenting the bacteria in a fermentation broth;   (b) disrupting the bacteria contained in the fermentation broth;   (c) cooling or maintaining the fermentation broth at a temperature of between about 0°-15° C.;   (e) adding nitric acid to the fermentation broth such that the fermentation broth obtains a pH of about 2.0;   (f) incubating the acidified fermentation broth at a temperature of between 0°-15° C. so as to kill the bacteria;   (g) raising the pH of the broth to about 8.5;   (h) disrupting and dispersing solids contained within the broth;   (i) centrifuging the fermentation broth so as to produce a supernatant liquid and inclusion body pellet containing inclusion bodies; and   (j) removing the supernatant liquid so as to obtain isolated inclusion bodies containing the heterologous protein.